One-Year Observation of the SCID-Repopulating Cell Activities of Human Cord Blood-Derived CD34-Positive and -Negative Hematopoietic Stem Cells
One-Year Observation of the SCID-Repopulating Cell Activities of Human Cord Blood-Derived CD34-Positive and -Negative Hematopoietic Stem Cells
复制标题
人脐带血 CD34 阳性和阴性造血干细胞 SCID 再生细胞活性的一年观察
DOI：
10.1007/s12015-019-09884-5
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发表时间：
2019
影响因子：
4.8
通讯作者：
Sonoda Yoshiaki
中科院分区：
文献类型：
--
作者：
Matsuoka Yoshikazu;Sumide Keisuke;Sonoda Yoshiaki
造血干细胞具有自我更新和多向分化潜能。因此，人HSC在整个生命过程中持续供应所有类型的成熟造血细胞。我们之前在人脐带血（CB）中发现了CD 34阴性（CD 34 −）造血干细胞（HSC）[1]。这些CD 34 − HSC的基因表达谱和分化潜力明显不同于CD 34阳性（CD 34+）HSC [2]。此外，CD 34 − HSC可以在体外和体内产生CD 34 + HSC [2]。因此，这两种CD 34+和CD 34 − HSC被认为代表了人类HSC层次中不同类别的原始HSC [2]。目前，重度联合免疫缺陷（SCID）-再生细胞（SRC）试验被认为是评估这些人HSC活性的金标准方法[3，4]。然而，这种SRC测定法对于精确分析实际人类长期HSC活性具有一些限制，因为免疫缺陷小鼠的寿命比野生型小鼠的寿命短。此外，辐射浪费受体小鼠，并进一步缩短这些免疫缺陷小鼠的寿命[5]。因此，难以在原代受体小鼠中随访人类造血细胞再增殖和HSC活性超过约20-30周。由于这些原因，需要连续移植以观察SRC活性超过30周。然而，只有一小部分存在于原代受体小鼠骨髓中的SRC可以转移到下一代受体小鼠中。因此，在连续移植期间大量SRC丢失。因此，在第二受体小鼠中重新增殖的人CD 45+细胞的百分比通常较小（图S1）。另一方面，在我们严格的饲养条件下，约50%的免疫缺陷受体小鼠存活50周，即使在照射后也是如此（表S1）。这些长寿命的辐照小鼠使我们能够评估人CB衍生的CD 34+和CD 34 − HSC活性一年，而无需连续移植。在这项研究中，我们首先从人CB中分离出18个谱系阴性（18 Lin −）CD 34 + CD 38 − CD 133 + GPI-80+和18 Lin − CD 34 − CD 133 +GPI-80+细胞，如先前报道的[2]（图S2）。在这些组分中，CD 34+和CD 34 − SRC的存在频率分别为1/4.9和1/8.1 [2]。然后，通过骨髓内注射将200个18 Lin − CD 34 + CD 38 − CD 133 +GPI-80+（41个CD 34 + SRC）和200个18 Lin − CD 34 − CD 133 +GPI-80+细胞（25个CD 34 − SRC）移植到经辐照（2.5戈伊）免疫缺陷（NOG或NSG）小鼠的左胫骨中[1，2]。在移植后20-50周，以大约10周的间隔分析人多谱系造血再生（包括CD 3 + T细胞、CD 19 + B细胞、CD 33+骨髓和CD 34+祖细胞）。所有接受CD 34+和CD 34 − SRCs的小鼠在移植后20周均显示出多谱系人类造血细胞的再增殖。在观察期间，CD 34+和CD 34 − SRC的再增殖率与人CD 45+细胞相当（图1a，B）。至于人CD 45+细胞再增殖，在任何时间点（20-50周），CD 34+和CD 34 − SRC之间均无显著差异（p> 0.05）。在植入CD 34+和CD 34 − SRC的两组中，小鼠骨髓中人CD 45+细胞的平均百分比在移植后20周达到峰值（分别为43.4%和42.2%），并逐渐下降，直到移植后50周。在第50周，小鼠骨髓中移植的人CD 45+细胞的平均百分比...
Hematopoietic stem cells (HSCs) inherently possess self-renewal activity and multi-lineage differentiation potential. Consequently, human HSCs continuously supply all types of mature hematopoietic cells throughout life. We previously discovered CD34-negative (CD34−) hematopoietic stem cells (HSCs) in human cord blood (CB)[1]. The gene expression profiles and differentiation potential of these CD34− HSCs clearly differ from those of CD34-positive (CD34+) HSCs [2]. Moreover, CD34− HSCs can generate CD34+ HSCs in vitro and in vivo [2]. Thus, these two CD34+ and CD34− HSCs were thought to represent a different class of primitive HSCs in the human HSC hierarchy [2]. Currently, the severe combined immunodeficiency (SCID)-repopulating cell (SRC) assay has been considered a gold standard method for the assessment of these human HSC activities [3, 4]. However, this SRC assay has some limitations for the precise analysis of actual human long-term HSC activity, since the lifetime of an immunodeficient mouse is shorter than that of a wild-type mouse. Moreover, irradiation wastes recipient mice and further shortens the life of these immunodeficient mice [5]. Thus, it is difficult to follow-up the human hematopoietic cell repopulation and HSC activity for more than approximately 20–30 weeks in the primary recipient mice. For these reasons, serial transplantation is required to observe SRC activity for more than 30 weeks. However, only a small portion of SRCs residing in the primary recipient mouse bone marrow can be transferred into next-generation recipient mouse. As a result, a large number of SRCs are lost during serial transplantation. Thus, the percentage of repopulating human CD45+ cells in the secondary recipient mice is generally small (Fig. S1). On the other hand, under our stringent breeding conditions, approximately 50% of immunodeficient recipient mice survive for 50 weeks, even after irradiation (Table S1). These long-lived irradiated mice enable us to assess human CB-derived CD34+ and CD34− HSC activities for one year without serial transplantation. In this study, we first isolated 18 Lineage-negative (18Lin−) CD34+ CD38− CD133+ GPI-80+ and 18Lin− CD34− CD133+GPI-80+ cells from human CB, as previously reported [2](Fig. S2). In these fractions, CD34+ and CD34− SRCs existed at a frequency of 1/4.9 and 1/8.1, respectively [2]. Then, 200 18Lin− CD34+CD38− CD133+GPI-80+(41 CD34+ SRCs) and 200 18Lin− CD34− CD133+GPI-80+ cells (25 CD34− SRCs) were transplanted into the left tibiae of irradiated (2.5 Gy) immunodeficient (NOG or NSG) mice by intra-bone marrow injection [1, 2]. Human multi-lineage hematopoietic repopulation (including CD3+ T-cells, CD19+ B-cells, CD33+ myeloid and CD34+ progenitor cells) was analyzed for 20–50 weeks after transplantation at intervals of approximately 10 weeks. All mice that received CD34+ and CD34− SRCs showed multi-lineage human hematopoietic cell repopulation at 20 weeks after transplantation. Both CD34+ and CD34− SRCs showed a repopulation rate that was comparable to that of human CD45+ cells during the observation period (Fig. 1a, b). As for human CD45+ cell repopulation, there was no significant difference between CD34+ and CD34− SRCs at any time point (20–50 weeks)(p> 0.05). In both groups engrafted with CD34+ and CD34− SRCs, the mean percentages of human CD45+ cells in the mouse bone marrow peaked at 20 weeks after transplantation (43.4 and 42.2%, respectively), and gradually decreased until 50 weeks after transplantation. At week 50, the mean percentages of human CD45+ cells in mouse bone marrow engrafted …
在非清髓性条件下移植造血干细胞的人源化 NOD/SCID/IL2Raamna(null) 小鼠显示出更长的寿命，并可对人类免疫缺陷病毒 1 型发病机制进行详细分析
DOI：
--
发表时间：
2007
期刊：
J Virol 81巻
影响因子：
--
作者：
齊藤健太;松田知己;原口徳子;永井健治;Watanabe S
通讯作者：
Watanabe S
影响因子：
7.8
作者：
Dick, J E
通讯作者：
Dick, J E